To facilitate an appreciation of the invention, this section may discuss the historical and technical background leading to the development of the invention, including observations, conclusions, and viewpoints that may be unique to an inventor. Accordingly, the background statements herein should not be construed as an admission regarding the content of the prior art.
A number of therapies have been developed to treat a variety of cancers. Many of these efforts have centered around chemotherapeutic regimens. In one particular combination chemotherapy regimen designed as a treatment for metastatic melanoma, response rates of 35-50% were reported with the “Dartmouth regimen” (a combination of DTIC, cisplatin, BCNU, and tamoxifen), but the duration of survival has remained at 6 to 10 months. High rates of remission also have been reported for aggressive high-dose intensity chemotherapy (Hryniuk et al., J. Clin. Oncol. Vol. 4, pp. 1162-1170 (1986)) and repletion of hematopoeisis with autologous bone marrow transplants. Nevertheless, the median duration of survival was short, approximately four months (Herzig, High-Dose Cancer Therapy: Pharmacology Hematopoietins, Stem Cells (Armitage and Antman, eds.), Williams and Wilkins (Baltimore), pp. 750-754 (1992)).
Significant improvements in survival on the order of several years have been noted in a small percentage of melanoma patients undergoing certain immunotherapies. Immunotherapies have included active specific immunotherapy with cancer vaccines, as well as the use of nonspecific boosters of the immune system, such as interleukin-2 (IL-2) and interferon-alpha (IFN-α) (Mitchell et al., “Specific Immunotherapy of Cancer with Vaccines”, Ann. N.Y. Acad. Sci. (Bystryn et al., eds.), pp. 153-166 (1993); Quan et al, “Principles of Biologic Therapy,” Cancer Treatment (Haskell, ed.) Philadelphia: W. B. Saunders (Philadelphia), pp. 57-69 (1995); Mitchell et al., J. Clin. Oncol., Vol. 12, pp. 402-411 (1994)). See also United States Patent Application Publication No. US 2003/0022820.
The identification of T-cell defined tumor antigens in melanoma has led to clinical trials that target cancer cells by attempting to augment the antigen-specific cellular immune response. This approach has been pursued in numerous vaccination strategies in which the antigens are delivered in an immunogenic context in an attempt to induce potent T cell responses in vivo. Although some clinical responses have been observed in the vaccine trials, the magnitude of the induced T-cell response has generally been low, or undetectable and correlated poorly with clinical responses. Immunization of melanoma patients with cancer antigens may increase the number of circulating CTL precursors; however it has not correlated with clinical tumor regression, suggesting a defect in function or activation in vivo.
Studies in mouse tumor models have demonstrated that adoptive immunotherapy, which involves in vitro immunization of T cells specific for one or more tumor antigens, may be efficacious with minimal toxicity. An obstacle in applying this strategy to the treatment of human tumors has been the identification of immunogenic antigens that render the tumor cells susceptible to CTL-mediated destruction. The isolation of tumor-reactive T cells from melanoma patients has led to the identification of some of the tumor antigens (epitopes) to which CTLs are directed. These include tyrosinase, MART-1/Melan A, gp 100, and MAGE. Of these, tyrosinase and MART-1 are nearly universally expressed on melanoma and therefore represent a desired target choice for adoptive immunotherapy (Van der Bruggen et al., Science, Vol. 254, pp. 1643-1647 (1991); Gaugler et al., J. Exp. Med. Vol. 179, pp. 921-930 (1994); Kawakami et al., J. Exp. Med., Vol. 180, 347-352 (1994); Brichard et al., J. Exp. Med., Vol. 178:489-495, 1993; Robbins et al., Cancer Res. 54:3124-3126, 1994; Bakker et al., J. Exp. Med. Vol. 179, pp. 1005-1009 (1994); Wolfel et al., Eur. J. Immunol. Vol. 24, pp. 759-764 (1994); and Visseren et al., J. Immunol. Vol. 154, pp. 3991-3998 (1995)).
Adoptive T cell therapy involves the removal of T cells from the host environment where tolerogenic mechanisms are active in vivo in cancer patients and contributes to the ineffective responses demonstrated in this patient population. CD8+ T cells may be stimulated ex vivo to generate antigen-specific CTLs (see, e.g., U.S. Pat. No. 6,225,042). Early adoptive immunotherapy approaches employed activated lymphocytes as a treatment for various cancers (Rubin et al., Biological Approaches to Cancer Treatment. Biomodulation (Mitchell, ed.), McGraw-Hill (New York), pp. 379-410 (1993)). Initially, lymphokine-activated killer cells (LAK), and later tumor-infiltrating lymphocytes (TIL), activated ex vivo with IL-2, were used, but the demonstration of efficacy was equivocal. These early controlled clinical trials failed to show an advantage to the use of the ex vivo-activated cells over the direct administration of IL-2 to melanoma patients. More recent studies by Yee et al. at Fred Hutchinson Cancer Research Center (Yee et al., PNAS, Vol. 99, pp. 16168-16173, (2002)) and Dudley et al. at NCI (Dudley et al., Science, Vol. 298, pp. 850-854 (2002)) have demonstrated the potential for certain adoptive T-cell therapeutic approaches. These studies involved use of either T-cell clones specific for MART-1 or gp100 and low-dose IL-2, or TILs expanded ex vivo with allogeneic feeder cells and high-dose IL-2. These studies confirmed that adoptive immunotherapy holds promise as a treatment of cancer, although its full development has been impeded by the lack of reproducible methods for ex vivo generation of therapeutic numbers of antigen-specific CD8+ CTLs (Oelke et al., Nat. Med., Vol. 9:619-624 (2003)).
Cytolytic, or cytotoxic, CD8+ T cells are a major line of defense against viral infections. CD8+ lymphocytes specifically recognize and lyse host cells that are infected with a virus. Although it would be desirable to harness the cytotoxic activity of CTLs, few in vitro/ex vivo procedures have been available to specifically activate CTLs. The identification of key melanoma-associated antigens and a method for specific in vitro activation of CTLs, allows for an efficient evaluation of adoptive immunotherapy for metastatic melanoma (see, in addition to Yee et al., Dudley et al., and Oelke et al., supra, and Leturcq et al., “Ex Vivo Generation of Potent Cytotoxic T Lymphocytes for the Treatment of Cancer: A Novel Antigen Presentation System”, Society of Biological Therapy 17th Annual Meeting, Abstract #40 (2002)).
While it is possible to use naturally occurring antigen presenting cells (APCs) for naïve T cell activation in vitro (e.g., dendritic cells, macrophages, B-cells, or autologous tumor cells), the efficiency of activation is low since the MHC molecules of native APCs contain many other peptide epitopes, thus allowing minimal presentation of selected epitopes. Most of these presented peptides represent normal, innocuous endogenous proteins. A more direct approach to this problem would be to activate CD8+ T cells specifically to those epitopes relevant to combating the disease.
One artificial APC which is an xAPC has been developed utilizing a Drosophila melanogaster (fruit fly) embryonic cell line, which expresses the major histocompatibility complex (MHC) Class I molecules (Leturcq et al., supra; Jackson et al., Proc. Natl. Acad. Sci. USA, Vol. 89, pp. 12117-12121 (1992); see also U.S. Pat. Nos. 6,225,042 and 6,355,479). Since Drosophila lacks an advanced immune system, Drosophila homologues to human TAP-1 and 2 peptide transporters, which are involved in the loading of peptide epitopes into the human MHC molecules, are absent. Hence, transfected Class I molecules and Class II molecules appear on the Drosophila cell surface as empty vessels. By incubating Drosophila cells transfected with MHC Class I- or MHC Class II-encoding expression vectors with exogenous synthetic peptides that bind to the specific MHC molecules (i.e., tumor antigen T-cell peptide epitopes), all of the available MHC molecules may be occupied with MHC-restricted, specific peptide epitope(s). In particular, the high density expression of MHC Class I molecules presenting single or multiple epitopes, and the addition of key co-stimulatory molecules B7-1 (CD80), CD70, LFA-3 (CD58), and ICAM-1 (CD54) on these Drosophila APCs, may permit the in vitro generation of potent, autologous cytotoxic CD8+ T cells which are specific for the selected peptides (Cai et al., Immunol. Rev., Vol. 165, pp, 249-265 (1998).
Various improvements in cell therapies have been developed. See, e.g., the following patent publications: U.S. Pat. Nos. 5,314,813, 5,529,921, 5,827,737, 6,001,365, 6,225,042, 6,251,627, 6,255,073, 6,362,001, 6,461,867, 6,790,662, and 6,828,150; WO 2002/065992 and 2002/092773; and EP 814,838. For example, three clinical studies (referred to as CTL-01, CTL-02, and CTL-03) in advanced malignant melanoma patients have been conducted where autologous CD8+ T cells are isolated from the patients, stimulated and expanded ex vivo, before being returned to the patients as antigen-specific cytotoxic T lymphocytes (CTLs). The ability to reproducibly generate potent antigen-specific CTLs ex vivo involves a primary stimulation with an embryonic Drosophila melanogaster cell line (SC2) that is transfected with human HLA class I, co-stimulatory and adhesion molecules which are important for optimum T cell activation. The transfected cells are used as artificial antigen presenting cells to stimulate naïve CD8+ T cells to drive them to effector cells with cytotoxic activity against target cells, which express the protein to which the CTLs were immunized against in vitro. Two different artificial APC lines have been used in these clinical studies. One expressing HLA-A2, B7.1 and ICAM (clone 666), and the other expressing these same three molecules, plus B7.2 and LFA-3 (clone 668).
A cell therapy product designated CTL-04, which is undergoing clinical investigation, has been developed with the Drosophila-based APCs. The cell therapy product is an autologous immunotherapeutic product prepared with ex vivo-activated autologous CD8+ CTLs exhibiting peptide specificity to up to six selected HLA-A2.1-restricted peptides from melanoma-associated antigens identified by the sequences, listed in SEQ ID NOS:5, 6, 7, 8, 9, and 70. The active component of the cell therapy product is the patient's own CD8+ cells, which have been activated ex vivo by exposure to selected peptide-loaded aAPCs having specificity for at least one of the six HLA-A2.1 restricted peptides listed in the SEQ ID NOS. provided above. These CTLs are: derived from autologous naïve T cells isolated from lymphapheresis samples harvested at a clinical site; primed ex vivo against melanoma antigenic peptide epitopes using artificial, inactivated Drosophila cells as the APCs; expanded by restimulation with autologous monocytes loaded with the melanoma antigenic epitopes in the presence of Interleukin-2 (IL-2) and Interleukin-7 (IL-7), followed by non-specific expansion using OKT®3; harvested, washed, and re-suspended in final formulation for infusion; and infused into the patient. The final product for re-infusion contains 1−10×109 CTL cells in 300 mL of Lactated Ringer's Injection Solution (76% v/v), 5% dextrose in normal saline (D5NS) (4% v/v), and human serum albumin (HSA) (20% v/v).
Of course, as with any drug, it is important to ensure safety and efficacy of the therapeutic product. Thus, before a cell therapy product such as CTL-04 is released for clinical use, it is typically subjected to various quality assurance tests. For example, the cell therapy product may be tested to confirm absence of Drosophila DNA by a PCR-based technique. Additionally, the product may be subjected to RT/PCR to confirm absence of known endogenous insect-specific RNA viruses, such as Drosophila Nodavirus (DrNV); Drosophila X virus (DXV), and Drosophila HPS-1-like virus. Furthermore, the BacT/Alert® may be used to test in-process and final product sterility. The sterility testing of cell products by the NIH Department of Transfusion Medicine for fungal, bacterial, and endotoxin content is mentioned in U.S. Patent Application Publication No. US 2006/0159667.
Notwithstanding the safety and efficacy of such cell therapies, there remains a desire to further develop cell therapies that are further assured as being safe and potent, especially in light of the FDA reclassifying such a cell therapy as a xenotransplantation product. This classification requires a separate set of guidelines, which includes specific mention of the drug as a xenotransplantation product, possible risk of zoonotic infections to both the recipient and close physical contacts, prevention of organ or blood donations after receiving the treatment, and the establishment of a long term monitoring program to determine if late toxicity occurs as a result of the therapy.